MiR-626 Targeting Keap1 To Activate Nrf2 Signaling Protects Retinal Pigment Epithelial Cells From Oxidative Damage

Background:Oxidative stimuli can lead to the over-production of reactive oxygen species（ROS）,including superoxide,hydroxyl radicals,and hydrogen peroxide（H2O2）,that can induce significant oxidative injury to retinal pigment epithelium（RPE）cells,retinal ganglion cells（RGCs）and other retinal cells,serving as a primary pathological mechanism of age-related macular degeneration（AMD）and other retinal degeneration diseases。In eukaryotic cells,nuclear-factor-E2-related factor 2（Nrf2）signaling represents the master anti-oxidant mechanism.Activation of the Nrf2 cascade,via pharmacological or genetic strategies,can efficiently protect RPE cells and other retinal cells from oxidative injury.A potential strategy to activate Nrf2 signaling is to inhibit/silence its suppressor protein Keap1.Methods:First the bioinformatics analyses were performed to explore potential Keap1-targeting miRNA:miR-626.In ARPE-19 cells,primary human RPE cells,human lens epithelial cells（HLECs）and primary human retinal ganglion cells（RGCs）,lentiviral constructs were utilized to exogenously over-express or inhibit miR-626expression.Afterwards,Western blotting（WB）,Quantitative real-time PCR（q PCR）were utilized to test miR-626 expression as well as expression and activation of Keap1-Nrf2 cascade genes.Cells were then treated with hydrogen peroxide（H₂O₂）to mimic oxidative injury,JC-1,TUNEL,Annexin V,CCK-8,Caspase-3 activity assays were applied to test mitochondrial depolarization,cell survival and apoptosis,as well as other cellular functions.Finally,plasma samples were collected from AMD patients and healthy donors,total RNAs were extracted,and expression of miR-626 was tested by q PCR assays.Results:Here,we indentified a novel kelch-like ECH-associated protein 1（Keap1）-targeting micro RNA,micro RNA-626（miR-626）that activates Nrf2 signaling.In ARPE-19 cells and primary human RPE cells,ectopic overexpression of miR-626targeting the 3′-UTR（3’-untranslated region）of Keap1 downregulated its expression,promoting Nrf2 protein stabilization and nuclear translocation,leading to expression of ARE-dependent genes（HO1,NOQ1 and GCLC）.Functional studies showed that miR-626 protected RPE cells from hydrogen peroxide（H2O2）-induced oxidative injury.Conversely,miR-626 inhibition induced Keap1 upregulation and Nrf2 cascade inhibition,exacerbating oxidative injury in RPE cells.Further studies demonstrated that miR-626 was ineffective in Keap1-knockout or Nrf2-knockout RPE cells.Importantly,miR-626 also activated Keap1-Nrf2 signaling cascade in human lens epithelial cells（HLECs）and primary human retinal ganglion cells（RGCs）,providing protection from H₂O₂.At last,we show that plasma miR-626 levels are significantly downregulated in age-related macular degeneration（AMD）patients than those in the healthy donors.Conclusions:The results of this study suggest that targeting Keap1 by miR-626protects RPE cells and other ophthalmic cells from oxidative injury via activation of Nrf2 signaling cascade.